The transcription factor p53 responds to variety of cellular stressors by inducing cell cycle arrest or apoptosis, playing a critical role in tumor suppression. Mutations in the p53 gene that compromise p53 functions occur in 50% of human cancers, and elevated levels of two p53 inhibitors Mdm2 and Mdm4 occur in most of the rest. Current dogma holds that Mdm2 mainly regulates p53 stability via its RING finger E3 ubiquitin ligase and Mdm4 mainly controls p53 transcriptional activity through concealing the p53 transcriptional activation domain. In vitro data have shown that Mdm2's RING E3 is also responsible for degradation of Mdm4 and itself, and a model is proposed that switch from Mdm2 degradation of p53 to self-degradation is responsible for p53 accumulation and activation after stress. Many p53 inducers including tumor suppressor p14ARF and ribosomal protein L11 stabilize and activate p53 through inhibition of Mdm2's E3 function. Thus, theoretically anticancer strategies targeting Mdm2 E3 function could cooperate with strategies targeting the Mdm2-p53 interaction to activate p53 in the millions of patients diagnosed with p53-positive cancers each year. Importantly however, detailed knowledge of the molecular mechanisms of Mdm2 E3 regulation will be required to achieve this goal. We have recently generated mice bearing a single-residue substitution in the Mdm2 RING finger domain abolishing its E3 function without affecting p53 binding. Unexpectedly however, in contrast to current notion our data have shown that 1) the Mdm2-p53 interaction, in the absence of Mdm2-mediated p53 ubiquitination, cannot control p53 activity, and 2) Mdm2 auto-ubiqutination is not the principle mechanism for Mdm2 degradation in vivo. Our analysis reveals yet another disconnect between hypotheses generated by in vitro transfection studies and mouse models. Based on this mouse model we will test three hypotheses: 1) Mdm2-Mdm4 interaction augments or necessitates Mdm2's E3 ligase function, 2) the binding of Mdm2 suppresses p53's apoptotic but not cell cycle arrest function, and 3) there is an unknown novel E3 ubiquitin ligase for Mdm2 degradation in vivo. Our specific aims are: Aim 1. To investigate the role of Mdm2 RING E3 in regulation of Mdm4 Aim 2. To investigate the non-redundant roles of Mdm2 and Mdm4 in regulation of p53. Aim 3. To investigate the role of Mdm2 in regulation of p53-induced cell cycle arrest and apoptosis. PUBLIC HEALTH RELEVANCE: Mutations in the tumor suppressor p53 gene that compromise p53 functions occur in 50% of human cancers, and elevated levels of two p53 inhibitors Mdm2 and Mdm4 occur in most of the rest. This project proposes to investigate the function and mechanism of the Mdm2- Mdm4-p53 regulatory loop using novel mouse models. Detailed knowledge of the molecular mechanisms and regulation of the Mdm2 E3 ubiqutin ligase is critically important for the design of future p53-based anticancer strategies.